Noxious chemical, thermal and mechanical stimuli excite peripheral nerve endings of small diameter sensory neurons (nociceptors) in sensory ganglia (e. g., dorsal root, nodose and trigeminal ganglia) and initate signals that are perceived as pain. These neurons are crucial for the detection of harmful or potentially harmful stimuli (heat) and tissue damage (H+ (local tissue acidosis), and/or stretch) which arise from changes in the extracellular space during inflammatory or ischaemic conditions (Wall, P. D., and Melzack, R., Textbook of Pain, 1994, New York: Churchill Livingstone).
Capsaicin, (N-[(4-hydroxy-3-methoxyphenyl)methyl]-8-methyl-6-nonenamide), the main pungent Ingredient in “hot” capsicum peppers, interacts with specific membrane recognition sites called vanilloid receptors (VR). These proteins are expressed almost exclusively by primary sensory neurons involved in nociception and neurogenic inflammation (Bevan, S., and Szolcsanyi, J., Sensory neuron-specific actions of capsaicin: mechanisms and applications, Trends Pharmacol. Sci., 1990, 11, 330-3). Capsaicin itself is a selective activator of thinly myelinated or unmyelinated nociceptive afferents (Szolcsanyi, J., Actions of capsaicin on sensory receptors, In Capsaicin Study, Pain, J. N. Wood, ed.: Academic, London, UK, 1993, pp. 1-26; and Szolcsanyi, J., Capsaicin-sensitive sensory nerve terminals with local and systemic efferent functions: facts and scopes of an unorthodox neuroregulatory mechanism, Prog. Brain Res., 1996, 113, 343-359).
Capsaicin and structurally-related derivatives show structure-function relationships and these effects can be blocked by a selective antagonist, capsazepine. The tricyclic diterpene resiniferatoxin (RTX) (Szolcsanyi, J., Szallasi, A., Szallasi, Z., Joo, F., and Blumberg, P. M., Resiniferatoxin: An ultrapotent neurotoxin of capsaicin-sensitive primary afferent neurons. Ann. N. Y. Acad. Sci., 1991, 632, 473-5) binds at the capsaicin binding site and has revealed a very localized distribution of capsaicin receptors at rat somatic and visceral primary sensory neurons (Szallasi, A., Nilsson, S., Farkas-Szallasi, T., Blumberg, P. M., Hoekfelt, T., and Lundberg, J. M., Vanilloid (capsaicin) receptors in the rat: distribution in the brain, regional differences in the spinal cord, axonal transport to the periphery, and depletion by systemic vanilloid treatment. Brain Res., 1995, 703, 175-83). Interestingly, the density of RTX receptor sites in nodose and dorsal root ganglia increased after ligation of the vagal and sciatic nerves (Szallasi et al., 1995).
Multiple effects of capsaicin on sensor neurons have been characterized (Szallasi, A. and Blumberg, P. M., Vanilloid receptor: new insights enhance potential as a therapeutic target, Pain, 1996, 68,195-208). Capsaicin excites the neuron resulting in calcium influx, the release of various neuropeptides such as CGRP and SP (Purkiss, J., Welch, M., Doward, S. and Foster, K, Capsaicin-stimulated release of substance P from culture dorsal root ganglion neurons: involvement of two distinct mechanisms, Biochem. Pharmacol., 2000, 59, 1403-1406) and cytokine release (Veronesi, B., Oortgiesen, M., Carter, J. D. and Devlin, R. B., Particulate matter initiates inflammatory cytokine release by activation of capsaicin and acid receptors in a human bronchial epithelial cell line, Toxicol. Appl. Pharmacol., 1999, 154,106-115). These events are believed to contribute to pain states and inflammation. Additionally, capsaicin and RTX can cause vanilloid receptor desensitization (Acs, G., Biro, T., Ace, P., Modarres, S. and Blumberg, P. M., Differential activation and desensitization of sensory neurons by resiniferatoxin, J. Neurosci., 1997, 17, 6522-5628) which, in animals, manifests as a state of reduced nociception. This phenomenon may constitute a novel approach to the treatment of neuropathic pain (Winter, J., Bevan, S. and Campbell, E. A., Capsaicin and pain mechanism, Br. J. Anaesth., 1995, 75, 157-168) and other pain states. However, under certain conditions, capsaicin and RTX can be neurotoxic (Bevan and Szolcsanyi, 1990).
Recently, one receptor protein for capsaicin was cloned from rat (Caterina, M. J., Schumacher, M. A., Tominaga, M., Rosen, T. A., Levine, J. D., and Julius, D., The capsaicin receptor: a heat-activated ion channel in the pain pathway, Nature (London), 1997, 389, 816-824) and shown to be a coincidence detector for H+ (low pH) and heat (Tominaga, M., Caterina, M. J., Malmberg, A. B., Rosen, T. A., Gilbert, H., Skinner, K., Raumann, B., Basbaum, A. I., and Julius, D., The cloned capsaicin receptor integrates multiple pain-producing stimuli, Neuron, 1998, 21, 531-543). VRI is a ligand-gated non-selective cation channel that shows pronounced outward rectification (Caterina et al., 1997). The vanilloid (“capsaicin”) receptor VR1 is activated by capsaicin and RTX, and activation of VRI is blocked by the antagonists capsazepine (CPZ) (Bevan, S., Hothi, S., Hughes, G., James, I. F., Rang, H. P., Shah, K., Walpole, C. S. J., and Yeats, J. C., Capsazepine: a competitive antagonist of the sensory neuron excitant capsaicin, Br. J. Pharmacol., 1992, 107, 544-52) and ruthenium red (RR) (Wood, J. N., Winter, J., James, I. F., Rang, H. P., Yeats, J., and Bevan, S., Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture, J. Neurosci., 1988, 8, 3208-20). VR1 Receptors are sensitive to vanilloids, heat and protons and thus these proteins are believed to be widely involved in the initiation, modulation and modification of pain (Woolf, C. J. and Salter, M. W., Neuronal plasticity: increasing the gain in pain, Science, 2000, 288, 1765-11768). Furthermore, VR1 is expressed in small nociceptive neurons of the dorsal root ganglion, consistent with its role in modulating peripheral pain (Tominaga, et al., 1998; and, Michael, G. J. and Priestley, J. V., Differential expression of the mRNA for the vanilloid receptor subtype 1 in calls of the adult rat dorsal root and nodose ganglia and its downreglulation by axotomy, J. Neurosci., 1999, 19, 1844-1854).
Patent application WO 99/09140 identifies the vanilloid receptor (VR) as a ligand-gated, non-selective cation channel that is expressed in sensory neurons and which is involved in nociceptive processes such as hyperalgesia. Resiniferatoxin, a naturally-occurring terpenoid phorbol derivative, binds to the VR protein with sub-nanomolar affinity and behaves as a functional agonist (stimulates calcium influx). Accordingly, iodinated and labeled RTX derivatives may similarly be potent in binding to the vanilloid receptor and may therefore be useful in identifying compounds that have affinity for vanilloid receptor when utilized in a receptor-binding assay. Indeed, tritiated-RTX is known to display a nanomolar affinity for VR, and can be used to identify or indicate compounds that also bind to this receptor.
Several [3H] RTX-based VR binding assays have been disclosed: a filtration assay (Szallasi, A. and Blumberg, P. M., Specific binding of resiniferatoxin, an ultrapotent capsaicin analog, by dorsal root ganglia membranes, Brain Res., 1990, 524, 106-111; and, Harvey, J. S., Davis, C., James, I. F. and Burgess, G. M., Activation of protein kinase C by the capsaicin analogue resiniferatoxin in sensory neurons, J. Neurochem., 1995, 65, 1309-1317) and a centrifugation assay (Szallasi, A., Goso, C., Blumberg, P. M., and Manzini, S., Competitive inhibition by capsazepine of [3H] resinferatoxin binding to central (spinal cord and dorsal root ganglia) and peripheral (urinary bladder and airways) vanilloid (capsaicin) receptors in the rat, J. Pharmacol. Exp. Ther., 1993, 267, 728-33; Szallasi, A. and Goso, C., Characterization by [3H] resiniferatoxin binding of a human vanilloid (capsaicin) receptor in post-morten spinal cord, Neurosci. Letters, 1994, 165, 101-104; and, Acs, G., Palkovits, M., and Blumberg, P. M., [3H] Resiniferatoxin binding by the human vanilloid (capsaicin) receptor, Mol. Brain Res., 1994, 23, 185-90). Use of filtration assay protocols is hampered by a high non-specific binding. Using the centrifugation assay, VRI receptors were found in several tissues, such as, dorsal root ganglia, spinal cord, dorsal vagal complex, trachea and main bronchi and urinary bladder. The affinities of [3H]RTX to the VR1 receptor were different with different tissues (Szallasi, A. and Blumberg, P. M., Vanilloid (capsaicin) receptors and mechanisms, Pharmacol. Rev., 1999, 51, 159-211). High expression of VR1 receptors was observed in human and rat spinal cord and dorsal root ganglia tissues (Szallasi, et al, 1993; Szallasi and Goso, 1994; and, Acs, et al., 1994). The affinity of [3H]RTX to VR1 receptors in rat spinal cord is higher than that in human spinal cord (Szallasi and Goso, 1994).
U.S. patent application 546,141 A0 to Blumberg, et. al., filed Apr. 15, 1991, describes labeled RTX or labeled congeners thereof, wherein representative radiolabels include tritium, 125iodine [125I] and 131iodine [311] having a preferred formula: 
Other representative congeners of RTX include those of the formula: wherein
n is 0 to 10 n is 0 to 10, preferably 2, and R′ is a fluorescent label. In lieu of radiolabeled RTX, the Blumberg application describes that biologically active congeners of RTX can be radiolabeled using the same synthetic route as for tritiated RTX, wherein such congeners have the general structure: wherein R is: R3 is OH, x is 0 or 1, R1, R2, R4 and R5 each are: H, OH, OC(═O)(CH2)nH, O(CH2)nH with n=0 to 10. In labeled compounds of the above congeners, at least of R1, R2, R4 or R5 is labeled, for instance, with 3H, 125I or 131I. In this formula, one of more of R1, R2, R4, and R5 is 3H. R3 should be OH so that the derivative is active. The preparation of 125I or 131I labeled RTX, tinyatoxin and congeners thereof possessing an unsubstituted m-hydrogen on the substituted phenylacetic acid side chain have been described, wherein such compounds can be prepared by iodination using chloramine T or using glucose oxidase or lactoperoxidase and, in the described compounds, one or more of R1, R2, R4 and R5 may be 125I or 131I.
PCT patent application WO 98/20867 to Blumberg, et. al., describes a pharmaceutical composition comprising a capsaicin agonist, a capsaicin antagonist and a pharmaceutically acceptable carrier wherein the capsaicin agonist is of the formula: wherein R1 is: R2 is hydroxy or methoxy; R3 is loweralkylaryl or an aryl group having 1 to 3 rings; and, R4 to R7, which may be the same or different, are independently selected from the group consisting of hydrogen, hydroxy, methoxy, sulfhydryl, nitro, amino, ethoxy, halo and OCOCH3.
No reference has heretofore disclosed a method for preparing an iodinated or labeled resiniferatoxin derivative. The object of the present invention is to provide a method for preparing an iodinated resiniferatoxin derivative and congeners thereof. Another object of the invention is to provide a method for preparing a labeled resiniferatoxin derivative and congeners thereof. Also another object of the invention is to provide a method for iodinating or labelling a specific site of a para-O-acetylated vanilloid receptor intermediate. A further object of the invention is to provide a method for use of an lodinated or labeled resiniferatoxin derivative and congeners thereof in a vanilloid receptor binding assay.